FimH mutants, compositions therewith and use thereof

ABSTRACT

Polypeptides comprising a FimH lectin domain comprising at least one an amino acid mutation that causes the FimH lectin domain to be in the low affinity conformation for mannose are described. Pharmaceutical compositions which comprise such polypeptides and methods of stimulating an immune response in a subject in need thereof by administration of the polypeptide are further described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to EP 21151126.6, filed Jan. 12, 2021,the disclosure of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “004852_12055_195US1_SeqList.txt” and a creation date of Jan.11, 2022, and having a size of 16 kb. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to the fields of medical microbiology andvaccines. In particular, the invention relates to polypeptidescomprising a FimH lectin domain comprising at least one amino acidmutation that causes the FimH lectin domain to be in a conformation withlow affinity for mannose and inducing high levels of antibody-mediatedinhibition of adhesion of E. coli to bladder epithelial cells uponadministration to a subject. Furthermore, the invention relates tocompositions which comprise such polypeptides and to methods ofstimulating an immune response in a subject in need thereof byadministration of the immunogenic polypeptide.

BACKGROUND OF THE INVENTION

Strains of E. coli responsible for extra-intestinal infections have beentermed extra-intestinal pathogenic E. coli (ExPEC). ExPEC are the mostcommon enteric Gram-negative organisms to cause extra-intestinalinfection in the ambulatory, long-term-care, and hospital settings.Typical extra-intestinal infections due to E. coli include urinary tractinfection (UTI), bacteremia, and sepsis. E. coli is a leading cause ofsevere sepsis and it is responsible for high morbidity and mortalityrates.

ExPEC, as other members of the Enterobacteriaceae family, produces typeI fimbriae, which aid in the attachment to mucosal epithelial surfaces.These type I fimbriae are hair-like structures which emanate from thesurface members of the Enterobacteriaceae family. The major component ofType I fimbriae is repeating subunits of FimA arranged in a right-handedhelix to form a filament approximately 1 μm in length and 7 nm indiameter with a central axial hole. Along with FimA as the majorsubunit, the fimbrial filament also contains FimF, FimG and FimH asminor protein subunits. The minor protein subunit FimH is amannan-binding adhesin that promotes adherence of Type I-fimbriatedbacteria to mannose-containing glycoproteins on eukaryotic cell surfacesand represents a family of proteins which bind to various targets,including mannan and fibronectin. Immune electron microscopy studieshave revealed that FimH is strategically placed at the distal tips ofType I fimbriae where it appears to be complexed with FimG, forming aflexible fibrillum structure, and is also placed longitudinally atvarious intervals along the filament.

The FimH adhesin protein has been shown to induce protection when usedas vaccine in various pre-clinical models against UTI (Langermann S, etal., 1997, Science, 276: 607-611; Langermann S, et al., 2000, J InfectDis, 181: 774-778; O'Brien VP et al., 2016, Nat Microbiol, 2:16196).

It has been shown that during E. coli infection, the lectin domain ofthe adhesin FimH, that binds to mannosylated receptors, can adopt twodistinct conformations: low-affinity for mannose (tense) andhigh-affinity for mannose (elongated/relaxed) (Kalas et al, 2017, SciAdv 10; 3(2)). The low affinity conformation promotes bacterial motilityand colonization of new tissues. The high-affinity conformation ensurestight bacterial adhesion under the mechanical forces of urine excretion.Furthermore, antibodies against low-affinity variant were shown to blockbacterial binding to uroepithelial cells and reduce CFU counts in thebladder (Tchesnokoca, 2011 Infect Immun. 79(10):3895-904; Kisiela, 2013Proc Natl Acad Sci, 19; 110(47):19089-94).

WO02102974 describes a number of FimH mutants that all comprise an aminoacid modification in the canyon region of the molecule. Specifically,WO02102974 describes variants wherein mannose interacting residues inthe binding pocket are mutated. This location of the mutation isselected because it would keep the FimH mutant in a more openconformation and thereby expose epitopes that are poorly accessible inthe wild-type protein. However, to date, to the best of our knowledge,none of these mutants have been further pursued as vaccine candidates.In clinical trials, only wild-type FimH has been used.

Thus, there remains a need in the art for vaccines that can inducehighly inhibitory antibodies against bacterial infections caused by E.coli.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides for a polypeptide comprising aFimH lectin domain comprising an amino acid other than phenylalanine (F)at the position corresponding to position 71 in the amino acid sequenceof SEQ ID NO: 1.

In a second aspect, the invention provides for a polypeptide comprisinga FimH lectin domain according to the first aspect wherein thepolypeptide further comprises an amino acid other than phenylalanine (F)at the position corresponding to position 144 in the amino acid sequenceof SEQ ID NO: 1.

In a third aspect, the invention provides a polynucleotide encoding apolypeptide according to the invention.

In a fourth aspect, the invention provides for a vector comprising thepolynucleotide according to the invention.

In a fifth aspect, the invention provides for a host cell comprising apolynucleotide or a vector according to the invention.

In a sixth aspect, the invention provides for a pharmaceuticalcomposition comprising a polypeptide, polynucleotide, or vectoraccording to the invention.

In a seventh aspect, the invention provides for a polypeptide accordingto the invention, a polynucleotide according to the invention, a vectoraccording to the invention, or a pharmaceutical composition according tothe invention for use in inducing an immune response against a bacteriumof the family of Enterobacteriaceae. The invention further relates to amethod for treating or preventing an enterobacillus-related condition asubject in need thereof the method comprising administering an effectiveamount of a polypeptide according to the invention, a polynucleotideaccording to the invention, a vector according to the invention, or apharmaceutical composition according to the invention.

In an eighth aspect the invention further provides for a method forproducing a polypeptide comprising expressing the polypeptide from arecombinant cell containing the polynucleotide of the invention and/orthe vector of the invention, optionally the method further comprisesrecovering the polypeptide which is optionally followed by formulationinto a pharmaceutical composition of the polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B: Functionality of antibodies induced by differentFimH variants. Wistar rats received 4 intramuscular immunizations at day0, 7, 10 and 18 with 60 ug/dose of the different FimH variants combinedwith a non-Freund's adjuvant (Speedy-rat model, Eurogentec). Serumsamples were obtained at day 0 (pre-immunization) and day 28(post-immunization).

FIG. 1A) Initial experiment with various FimH variants (indicated undergraph). Inhibitory antibody titers (IC50) were calculated based on a4-parameter logistic regression model fitted on a 12-step dilutioncurve. Data represents mean of duplicate serum samples from 2animals/group.

FIG. 1B) Separate experiment with FimH mutants F144V, F71Y, andF144V/F71Y double mutant. Inhibitory antibody titers (IC50) werecalculated based on a 4-parameter logistic regression model fitted on a6-step dilution curve. The graph shows individual IC50 titerspre-immunization and post-immunization of serum samples measured induplicate and the GMT (geometric mean titer)±95% CI (confidenceinterval). LOD: limit of detection.

FIG. 2A and FIG. 2B: Conformational state of the different FimH lectindomain variants in the presence and absence of the mannoside ligand asdetermined by NMR spectroscopy. FIG. 2A shows ¹⁵N HSQC NMR spectra ofuniformly ¹⁵N-labeled FimH_(LD) variants in the low affinity state (L)when no mannoside ligand is bound (e.g. the apo state) and FIG. 2B showsthe spectra in the high affinity state (H) when the mannoside ligand isbound (e.g. the ligand state). The key amino acid residues that undergoa chemical shift upon binding of the mannoside ligand are indicated inboxes. The residues have been identified from publically available NMRspectra from E. coli K12 (Rabbani S et al, J Biol. Chem., 2018,293(5):1835-1849) except for residue number 1 and 2, which were specificfor E. coli 23-10, indicating that the wild type FimH_(LD) 23-10 proteinhas a slightly different conformation in the apo state compared toFimH_(LD) of E. coli K12.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for a novel polypeptide comprising a FimH lectindomain wherein the FimH lectin domain is “locked” in a conformation withlow affinity for mannose, also referred to herein as the ‘low affinityconformation’. The present invention is based in part on the observationthat FimH antigen in the low affinity for mannose conformation iscapable of inducing antibodies that can inhibit mannoside-mediatedadhesion. These antibodies are highly inhibitory and have an enhancedeffect in preventing or treating bacterial infections. It was foundherein that a FimH lectin domain with an F71Y mutation has asurprisingly good combination of desirable properties that for instancemakes it very suitable for use in vaccines against UTI, e.g. to preventor reduce recurrent UTI.

Accordingly, in a first aspect, the invention provides for apolypeptide, preferably an immunogenic polypeptide, comprising a FimHlectin domain, comprising an amino acid other than phenylalanine (F) ata position corresponding to position 71 in the reference amino acidsequence of SEQ ID NO: 1.

In a second aspect, the invention further provides for a polypeptide,preferably an immunogenic polypeptide, comprising a FimH lectin domain,comprising an amino acid other than phenylalanine (F) at both positionscorresponding to positions 71 and 144, respectively, in the referenceamino acid sequence of SEQ ID NO: 1. This “double-mutant” remains lockedin the low affinity conformation and is thus equally capable of inducingantibodies that can inhibit mannoside-mediated adhesion. In addition toinducing these inhibitory antibodies, it was found herein that the F71Yand F144V double mutant of the FimH lectin domain is unable to switchback to the high affinity conformation which gives the double mutant asurprisingly high stability. This inability to switch back to the highaffinity conformation is a very desirable property that makes thedouble-mutant very suitable for instance for use in vaccines againstUTI, e.g. to prevent or reduce recurrent UTI, as it inter alia ensuresthat no additional quality or stability controls are needed to testwhether the conformation is stable during storage or under otherstorage- or use conditions.

The amino acid positions 71 and 144 as used herein refer to positions 71and 144, respectively, in the reference amino acid sequence of the FimHlectin domain of SEQ ID NO: 1. In amino acid sequences of the inventionother than SEQ ID NO: 1, preferably, the amino acid positions 71 and 144are present at a position in that other amino acid sequence thatcorrespond to the positions 71 and 144, respectively, in SEQ ID NO: 1,in a sequence alignment, preferably in a ClustalW (1.83) sequencealignment using default settings. The skilled person will know how toidentify corresponding amino acid positions in FimH lectin domain aminoacid sequences other than SEQ ID NO: 1 using amino acid sequencealignment algorithms as defined hereinabove.

Throughout the application, a polypeptide of the invention comprising aFimH lectin domain that comprises an amino acid other than phenylalanine(F) at the position corresponding to position 71 in the amino acidsequence of SEQ ID NO:1 will be referred to herein as “FimH(F71mut)”.Likewise, a polypeptide of the invention comprising a FimH lectin domainthat comprises an amino acid other than phenylalanine (F) at both thepositions corresponding to positions 71 and 144, respectively, in theamino acid sequence of SEQ ID NO:1 will be referred to herein as“FimH(F71mut/F144mut)”.

In certain embodiments, FimH(F71mut) comprises an amino acid selectedfrom the group of tyrosine (Y) and tryptophan (W) at the position thatcorresponds to position 71 in SEQ ID NO: 1.

In certain embodiments, FimH(F71mut) comprises Tyrosine (Y) at theposition that corresponds to position 71 in SEQ ID NO: 1.

In certain embodiments, the FimH(F71mut/F144mut) comprises an amino acidselected from the group consisting of valine (V), isoleucine (I),leucine (L), glycine (G), methionine (M), and alanine (A) at theposition that corresponds to position 144 in SEQ ID NO: 1.

In certain embodiments, the FimH(F71mut/F144mut) comprises Valine (V) atthe position that corresponds to position 144 in SEQ ID NO: 1.

In certain embodiments, at least one of the FimH(F71mut) and theFimH(F71mut/F144mut) lectin domains have an amino acid sequence havingat least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with SEQ ID NO: 1.

In one embodiment, the FimH(F71mut) or FimH(F71mut/F144mut) comprise atleast one mutation that causes the FimH lectin domain to be in the lowaffinity for mannose conformation. In one embodiment, the FimH(F71mut)has been mutated at a position that corresponds to position 71 in SEQ IDNO: 1. In one embodiment, FimH(F71mut/F144mut) has been mutated at thepositions that corresponds to positions 71 and 144 in SEQ ID NO: 1.

A FimH lectin domain with low affinity for mannose in this context isdefined herein as a FimH lectin domain binding to a mannoside ligandwith a dissociation constant (K_(D)) of at least 1000 nM or higher, asmeasured using surface plasmon resonance, e.g. under the conditionsspecified in example 2. A FimH lectin domain with high affinity formannose is defined herein as a FimH lectin domain binding to a mannosideligand with a K_(D) that is 100 nM or lower, under the same conditions;typically wild-type FimH falls in this category. FimH lectin domainswith a K_(D) between 100 nM and 1000 nM under these conditions isreferred to herein as having intermediate affinity for mannose.

The terms ‘mutant’, ‘mutation’, ‘mutated’ or ‘substitution’,‘substituted’ in this context mean that another amino acid is present onthe indicated position than in the corresponding parent molecule, whichhere is a polypeptide comprising a FimH lectin domain with F atpositions 71 and 144. Such parent molecule may exist physically aspolypeptide or in the form of nucleic acid encoding such polypeptide,but may also merely exist in silico or on paper as amino acid sequenceor a corresponding nucleic acid sequence encoding the amino acidsequence. A mutation or substitution in this context is therefore alsoconsidered present for instance if a protein is expressed from a nucleicacid that has been synthesized such that it encodes the mutation orsubstitution, even though the nucleic acid encoding the correspondingparent molecule was not initially actually prepared during the process,e.g. when the nucleic acid molecule has been prepared entirely bychemical synthesis.

Preferably, the mutation is a substitution of a single amino acidresidue. The mutation is preferably a substitution of an amino acidresidue that corresponds to at least one of the positions 71 and 144 inSEQ ID NO: 1, respectively. Preferably, the mutation is a substitutionof a phenylalanine (F) by another amino acid at a position correspondingto position 71 in SEQ ID NO: 1. Preferably the polypeptide of theinvention further comprises a substitution of a phenylalanine (F) byanother amino acid at a position corresponding to position 144 in SEQ IDNO: 1. In FimH(F71mut), the position that corresponds to position 71 inSEQ ID NO: 1 is preferably substituted for an amino acid selected fromthe group consisting of tyrosine (Y) and tryptophan (W). In onepreferred embodiment, FimH(F71mut) comprises the substitution ofphenylalanine (F) to tyrosine (Y) at position 71. In certainembodiments, FimH(F71mut) is a non-naturally occurring polypeptide whichcomprises a tyrosine at position 71. In certain embodiments,FimH(F71mut) has a tyrosine at position 71 instead of the naturallyoccurring phenylalanine (referred to as “FimH(F71Y)”). In certainembodiments, FimH(F71mut) has a tyrosine at position 71 instead of anaturally occurring amino acid other than phenylalanine (‘FimH(x71Y)’,wherein x would be an amino acid other than phenylalanine or tyrosine ina parent molecule).

In one embodiment, FimH(F71mut/F144mut) comprises a mutation at thepositions that corresponds to positions 71 and 144 in SEQ ID NO: 1. Themutation at position 71 is preferably a substitution as describedherein. The mutation at position 144 is preferably a substitution of anamino acid residue that corresponds to position 144 in SEQ ID NO: 1.Preferably, the mutation is a substitution of a phenylalanine (F) aminoacid residue at a position corresponding to position 144 in SEQ IDNO: 1. Preferably, the amino acid at position 144 is substituted by anamino acid selected from the group consisting of valine (V), isoleucine(I), leucine (L), glycine (G), methionine (M), and alanine (A). In onepreferred embodiment, FimH(F71mut/F144mut) comprises the substitution ofphenylalanine (F) to valine (V) at position 144.

In certain embodiments FimH(F71mut/F144mut) is a non-naturally occurringpolypeptide which comprises a tyrosine at position 71 and a valine atposition 144. In certain embodiments, FimH(F71mut/F144mut) has atyrosine at position 71 instead of the naturally occurring phenyalanineand a valine at position 144 instead of the naturally occurringphenylalanine. In certain embodiments, FimH(F71mut/F144mut) has atyrosine at position 71 instead of a naturally occurring amino acidother than phenylalanine as well as a valine at position 144 instead ofthe naturally occurring amino acid other than phenylalanine.

Full-length FimH (FimH_(FL)) is composed of two domains: the N-terminallectin domain (FimH_(LD)) connected to the C-terminal pilin domain(FimH_(PD)) by a short tetra-peptides loop linker. In certainembodiments of the invention, the polypeptide comprising the FimH lectindomain according to the invention does not comprise a FimH pilin domain.In another embodiment of the invention, the polypeptide comprising theFimH lectin domain according to the invention further comprises a FimHpilin domain. In one embodiment, FimH(F71mut) or FimH(F71mut/F144mut) isa full length FimH polypeptide wherein the FimH lectin domain comprisesan amino acid sequence as herein defined above for FimH(F71mut) andFimH(F71mut/F144mut), respectively. In certain embodiments of theinvention, the polypeptide comprising the FimH lectin domain accordingto the invention is a fusion polypeptide of the FimH lectin domain fusedto another polypeptide, which other polypeptide may be any polypeptideof interest, and this needs neither to be related to FimH nor to beassociated with FimH in nature. In certain embodiments, the polypeptidecomprising the FimH lectin domain of the invention is a fusionpolypeptide that further comprises a FimH pilin domain and in additioncomprises another polypeptide, which other polypeptide may be anypolypeptide of interest, and this needs neither to be related to FimHnor to be associated with FimH in nature. The fusion polypeptides of theinvention can for instance also be used as immunogens for vaccinationpurposes.

In one embodiment, the FimH_(FL) polypeptide comprises an amino acidsequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2, whereby,preferably the positions 71 or 71 and 144 comprise amino acid residuesas herein defined above for FimH(F71mut) and FimH(F71mut/F144mut),respectively. In certain embodiments, the FimH_(FL) polypeptide maycomprise a sequence having SEQ ID NO: 2, with the exception of the F71Yand optionally the F144V substitution as described herein. In anotherembodiment, the polypeptide is a FimH_(FL) having at least about 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with SEQ ID NO: 4 or preferably at least with amino acids22-300 thereof, whereby, the positions 71 or 71 and 144 comprise aminoacid residues as herein defined above for FimH(F71mut) andFimH(F71mut/F144mut), respectively. In certain embodiments, theFimH_(FL) polypeptide may comprise a sequence having SEQ ID NO: 4 or atleast amino acids 22-300 thereof, with the exception of the F71Y and/orthe F144V substitution as described herein.

In certain embodiments, the FimH_(FL) polypeptide comprises an aminoacid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 23-45 and55 as described in U.S. Pat. No. 6,737,063, which is incorporated hereinin its entirety, whereby, the positions 71 or 71 and 144 comprise aminoacid residues as herein defined above for FimH(F71mut) andFimH(F71mut/F144mut), respectively.

FimH polypeptides are highly conserved between various strains of E.coli, and they are also highly conserved among a wide range ofgram-negative bacteria. Moreover, the amino acid changes that occurbetween strains generally occur at similar amino acid positions. As aresult of the high conservation of FimH between E. coli strains, FimHpolypeptides from one strain are capable of inducing antibody responsesthat inhibit or prevent other E. coli strains from binding to cells by aFimH lectin and/or provide protection and/or treatment against infectioncaused by other E. coli strains. Accordingly, in one embodiment theFimH_(FL) polypeptide comprises an amino acid sequence that has at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity withSEQ ID NO: 5, or preferably at least with amino acids 22-300 thereof,whereby, the positions 71 or 71 and 144 comprise amino acid residues asherein defined above for FimH(F71mut) and FimH(F71mut/F144mut),respectively. In certain embodiments, the FimH_(FL) polypeptide maycomprise a sequence having SEQ ID NO: 5 or at least amino acids 22-300thereof, with the exception of the F71Y substitution and optionally theF144V substitution as described herein. In another embodiment, theFimH_(FL) polypeptide comprises an amino acid sequence that has at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity withSEQ ID NO: 6, whereby, the positions 71 or 71 and 144 comprise aminoacid residues as herein defined above for FimH(F71mut) andFimH(F71mut/F144mut), respectively. In certain embodiments, theFimH_(FL) polypeptide may comprise a sequence having SEQ ID NO: 6, withthe exception of the F71Y substitution, and optionally the F144Vsubstitution, as described herein.

In certain embodiments, FimH preferably is E. coli FimH.

As used herein, the term “periplasmic chaperone” is defined as a proteinlocalized in the periplasm of bacteria that is capable of formingcomplexes with a variety of chaperone-binding proteins via recognitionof a common binding epitope (or epitopes). Chaperones serve as templatesupon which proteins exported from the bacterial cell into the periplasmfold into their native conformations. Association of thechaperone-binding protein with the chaperone also serves to protect thebinding proteins from degradation by proteases localized within theperiplasm, increases their solubility in aqueous solution, and leads totheir sequentially correct incorporation into an assembling pilus.Chaperone proteins are a class of proteins in gram-negative bacteriathat are involved in the assembly of pili by mediating such assembly,but are not incorporated into the structure. FimC is the periplasmicchaperone protein of FimH. A FimC polypeptide for use in the instantinvention has an amino acid sequence having at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or about 100% sequence identity with SEQ IDNO: 3. In certain embodiments, the FimC polypeptide has an amino acidsequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or about 100% sequence identity with SEQ ID NO: 29 as described inU.S. Pat. No. 6,737,063, which is incorporated herein in its entirety.The non-covalent complex of FimC and FimH is named FimCH.

Accordingly, in a further aspect, the invention provides for a complexcomprising a polypeptide comprising a FimH(F71mut) orFimH(F71mut/F144mut) as defined herein and further comprising a FimHpilin domain or a full length FimH as defined herein, and a FimCpolypeptide as defined herein.

In one embodiment of the invention, the FimH(F71mut) orFimH(F71mut/F144mut) lectin domains are part of a polypeptide furthercomprising a FimH pilin domain, which polypeptide is complexed with FimCto form a FimCH complex.

The inventors of the present application have created several FimHlectin domain variants with different amino acid changes and tested themfor efficiency in various assays (see the examples).

The herein described FimH(F71mut) and FimH(F71mut/F144mut) were bothcapable of forming FimCH complex.

In one embodiment, the FimH(F71mut) or FimH(F71mut/F144mut) lectindomains are part of a FimH_(FL) polypeptide having at least about 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:2, which optionally is complexed with a FimC polypeptide to form a FimCHcomplex. A FimH_(FL) polypeptide in its final form, i.e. a matureFimH_(FL) polypeptide does typically not include the signal peptide,which is for instance shown as amino acids 1-21 of SEQ ID NOs: 4 and 5,i.e. a mature FimH_(FL) polypeptide of SEQ ID NO: 4 or SEQ ID NO: 5 isunderstood to include amino acids 22-300 of these sequences whiletypically lacking amino acids 1-21 thereof. For recombinant productionof a FimH_(FL) polypeptide it is useful to encode a mature FimH_(FL)polypeptide that includes the signal peptide in the recombinant hostcell, to get transport across the inner (cytoplasmic) membrane via thegeneral secretory pathway leading to periplasmic location of thepolypeptide (sometimes referred to as ‘periplasmic expression’), but inthe final mature FimH_(FL) polypeptide as isolated and for instance usedin pharmaceutical compositions, the signal peptide typically is nolonger present as a result of processing by the recombinant cell that isexpressing the polypeptide.

In one embodiment, the FimCH complex comprises or consists of a FimCprotein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at leastabout 99%, or 100% sequence identity with SEQ ID NO: 3 and a FimHprotein comprising a FimH(F71mut) or FimH(F71mut/F144mut) lectin domainas herein defined above. In certain embodiments, the FimCH complexcomprises or consists of a FimC protein having at least 80%, 85%, 90%,95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identitywith SEQ ID NO: 3 and a FimH protein or a FimH_(FL) protein thatcomprises FimH(F71mut) or FimH(F71mut/F144mut) lectin domain as hereindefined above, whereby the FimH_(FL), protein preferably comprises a F92substitution (e.g. in SEQ ID NOs: 4 or 5 that still include the signalpeptide). Optionally, the FimCH complex comprises or consists of a FimCprotein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at leastabout 99%, or 100% sequence identity with SEQ ID NO: 3 and a FimHprotein that comprises a F71(Y) and a F144(V) substitution in the lectindomain or a full-length FimH protein that comprises a F92(Y) and aF165(V) substitution (e.g. in SEQ ID NOs: 4 or 5 that still include thesignal peptide)

In a full length FimH that would still include the signal peptide aminoacid position 92 corresponds to amino acid position 71 and position 165corresponds to amino acid position 144 in the FimH lectin domain. Theskilled person will know how to identify corresponding amino acidpositions in full length FimH amino acid sequences and in FimH lectindomain amino acid sequences using amino acid sequence alignmentalgorithms as defined hereinabove.

In one embodiment, complexes comprising the E. coli chaperone FimC andpolypeptides comprising FimH(F71mut) or FimH(F71mut/F144mut) may beformed by co-expressing the FimH(F71mut)- orFimH(F71mut/F144mut)-comprising polypeptides along with FimC, from arecombinant cell.

In one embodiment, the FimCH complex comprises a FimC originating fromone bacterial strain while FimH originates from a different bacterialstrain. In another embodiment, the FimCH complex comprises a FimC and aFimH both originating from the same bacterial strain. In certainembodiments, FimH or FimC or both FimH and FimC may be artificialsequences not from actual bacterial isolates that exist in nature, e.g.they can also be based upon consensus sequences or combinations ofnatural isolates.

In one embodiment, the FimCH complex comprises at least one polypeptidethat comprises a His-tag. In one embodiment, the full-length FimH asdescribed herein comprises a His-tag or the FimC as described hereincomprises a His-tag. Preferably, in the FimCH complex, the FimCcomprises the His-tag. A His-tag as used herein is a stretch ofhistidine (His) residues, e.g. six His residues, which may be addedinternally or preferably at the N- or C-terminus of a protein. Such atag has well-known use for ease of purification.

In a further aspect, the invention pertains to a polynucleotide encodinga polypeptide comprising a FimH(F71mut) or FimH(F71mut/F144mut) lectindomain as defined herein above. The polynucleotide may be preceded by apromoter operably linked thereto. In certain embodiments, the promoteris endogenous to the FimH coding sequence. In certain embodiments, thepromotor is an endogenous promoter driving the expression of FimH in abacterium of the Enterobacteriaceae family. In other embodiments, thepromoter is heterologous to the FimH coding sequence, e.g. a strongpromoter known to the skilled person for use in recombinant expressionsystems is used. For example, a pET-DUET vector comprising an inducibleLac promoter can be used for expression of a FimH(F71mut) orFimH(F71mut/F144mut) polypeptide of the invention and/or for expressionof a FimC polypeptide. In case of an inducible promotor such as a Lacpromoter or Tac promoter, IPTG can be used to induce expression.Preferably, the polynucleotide is isolated from its natural environment.In certain embodiments, the invention provides an isolatedpolynucleotide according to the invention. The polypeptide can be arecombinant, synthetic or artificial polynucleotide. The polynucleotidemay be in any form of nucleic acid, e.g. DNA or RNA, preferably DNA. Thepolynucleotide may comprise one or more nucleotides that are not presentin a naturally occurring FimH encoding polynucleotide. Preferably, thepolynucleotide has one or more nucleotides that are not present in anaturally occurring FimH-encoding polynucleotide at its 5′-end and/or3′-end. The sequences of the encoded mature FimC and/or FimH maypreferably be preceded by a signal peptide in the polypeptides asencoded by the respective polynucleotides, and the signal peptides maybe endogenous signal peptides to the FimC and/or FimH polypeptides (i.e.signal peptides as occurring in nature for these proteins) respectively,or they may be heterologous signal peptides, i.e. signal peptides fromother proteins or synthetic signal peptides. The signal peptides areuseful for periplasmic expression, but are typically cleaved off and notpresent in the finally produced and purified FimC and/or FimH,respectively.

In a further aspect, the invention relates to vector comprising apolynucleotide encoding a FimH(F71mut) or FimH(F71mut/F144mut)polypeptide of the invention. In certain embodiments, the vector is aplasmid or a viral vector, preferably a plasmid. The vector ispreferably in the form of DNA, e.g. a DNA plasmid. In certainembodiments, the vector comprises the polynucleotide of the inventionoperably linked to a promoter, meaning that the polynucleotide is undercontrol of a promoter. The promoter may be located upstream of thepolynucleotide that encodes the polypeptide of the invention, e.g. in anexpression cassette in a plasmid.

In yet a further aspect, the invention relates to a host cell comprisinga polynucleotide encoding a FimH(F71mut) or FimH(F71mut/F144mut)polypeptide of the invention or a vector as described herein. Thepolynucleotide encoding the FimH(F71mut) or FimH(F71mut/F144mut)polypeptide of the invention can be introduced to the cell by commonmolecular biology methods. Such nucleic acid may be extrachromosomal,e.g. on a plasmid or other vector, or it may be integrated into thegenome of the host cell. Preferably the nucleic acid encoding a proteinis operably coupled to a sequence driving expression of the nucleic acidin the host cell, such as a promoter. The promoter may be a constitutivepromoter, or it may be a promoter of which the activity can beregulated, e.g. repressed or induced upon certain conditions, e.g.temperature changes or presence of certain chemicals or proteins in thecell, all of which are as such well known in the art.

The host cell may be an isolated cell. The host cell may be cultured ina culture medium, e.g. in a culture vessel such as a bioreactor. Thecell may be any microbial, prokaryotic or eukaryotic cell, which issuitable for expression of the polynucleotide encoding the FimH(F71mut)or FimH(F71mut/F144mut) polypeptide of the invention. Preferably, thehost cell is bacterial host cell. Preferably the bacterial host cell isa gram-negative bacterial cell. Preferably the host cell is selectedfrom E. coli and Klebsiella. Preferably, the host cell is E. coli.

If required and/or if desired, a FimH(F71mut) or FimH(F71mut/F144mut)polypeptide of the invention or the polynucleotide encoding aFimH(F71mut) or FimH(F71mut/F144mut) polypeptide of the invention can beincorporated into a pharmaceutically active mixture by adding apharmaceutically acceptable carrier.

Accordingly, in a further aspect, the invention also provides for acomposition, preferably a pharmaceutical composition comprising aFimH(F71mut) or FimH(F71mut/F144mut) polypeptide of the invention or apolynucleotide encoding a FimH(F71mut) or FimH(F71mut/F144mut)polypeptide of the invention.

The (pharmaceutical) compositions of the invention may comprise anypharmaceutically acceptable excipient including a carrier, filler,preservative, solubilizer and/or diluent. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Examples of suitable pharmaceutical carriers are known andfor instance described in textbooks and manuals.

In certain embodiments, the compositions of the invention additionallycomprise one or more buffers, e.g., Tris-buffered saline, phosphatebuffer, HEPES, or sucrose phosphate glutamate buffer.

In certain embodiments, the compositions of the invention additionallycomprise one or more salts, e.g., Tris-hydrochloride, sodium chloride,calcium chloride, potassium chloride, sodium phosphate, monosodiumglutamate, and aluminum salts (e.g., aluminum hydroxide, aluminumphosphate, potassium aluminum sulfate, or a mixture of such aluminumsalts).

The compositions of the invention can be used for eliciting an immuneresponse in a host to whom the composition is administered, i.e., areimmunogenic. Thus, the compositions of the invention can be used asvaccines against an infection caused by a bacterium of the family ofEnterobacteriaceae, preferably against an infection caused by Klebsiellaor E. coli, more preferably E. coli and can thus may optionally compriseany additional components suitable for use in a vaccine. For example, anadditional optional component of a vaccine composition is an adjuvant asdescribed herein.

In certain embodiments, the compositions of the invention additionallycomprise a preservative, such as phenol, benzethonium chloride,2-phenoxyethanol, or thimerosal. In a specific embodiment, the(pharmaceutical) compositions of the invention comprise 0.001% to 0.01%preservative. In other embodiments, the (pharmaceutical) compositions ofthe invention do not comprise a preservative.

In certain embodiments, the compositions of the invention are formulatedto be suitable for the intended route of administration to a subject.For example, the compositions of the invention can be formulated to besuitable for subcutaneous, parenteral, oral, intradermal, transdermal,colorectal, intraperitoneal, intravaginal, or rectal administration. Ina specific embodiment, the pharmaceutical composition can be formulatedfor intravenous, oral, buccal, intraperitoneal, intranasal,intratracheal, subcutaneous, intramuscular, topical, intradermal,transdermal or pulmonary administration, preferably intramuscularadministration.

The compositions of the invention can be included in a container, pack,or dispenser together with instructions for administration.

In certain embodiments, the compositions of the invention can be storedbefore use, e.g., the compositions can be stored frozen (e.g., at about−20° C. or at about −70° C.); stored in refrigerated conditions (e.g.,at about 2-8° C., e.g. about 4° C.); or stored at room temperature.

In an embodiment, the pharmaceutical composition of the invention hereinfurther comprises an adjuvant. As used herein, the term “adjuvant”refers to a compound that when administered in conjunction with or aspart of a composition of the invention augments, enhances and/or booststhe immune response to FimH, but when the adjuvant compound isadministered alone does not generate an immune response to the conjugateand/or FimH. Adjuvants can enhance an immune response by severalmechanisms including, e.g., lymphocyte recruitment, stimulation of Band/or T cells, and stimulation of antigen presenting cells.

In certain embodiments, the pharmaceutical compositions of the inventioncomprise, or are administered in combination with, an adjuvant. Theadjuvant for administration in combination with a composition of theinvention can be administered before, concomitantly with, or afteradministration of the immunogenic compositions. In certain embodiments,FimH(F71mut) or FimH(F71mut/F144mut) and the adjuvant are administeredin the form of a single composition.

In other embodiments, the pharmaceutical compositions of the inventiondo not comprise, and are not administered in combination with, anadjuvant.

Specific examples of adjuvants include, but are not limited to, aluminumsalts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, and aluminum oxide, including nanoparticles comprising alum ornanoalum formulations), calcium phosphate (e.g. Masson J D et al, 2017,Expert Rev Vaccines 16: 289-299), monophosphoryl lipid A (MPL) or3-de-O-acylated monophosphoryl lipid A (3D-MPL) (see e.g., UnitedKingdom Patent GB2220211, EP0971739, EP1194166, U.S. Pat. No.6,491,919), AS01, AS02, AS03 and AS04 (all GlaxoSmithKline; see e.g.EP1126876, U.S. Pat. No. 7,357,936 for AS04, EP0671948, EP0761231, U.S.Pat. No. 5,750,110 for AS02), imidazopyridine compounds (seeWO2007/109812), imidazoquinoxaline compounds (see WO2007/109813),delta-inulin (e.g. Petrovsky N and PD Cooper, 2015, Vaccine 33:5920-5926), STING-activating synthetic cyclic-di-nucleotides (e.g.US20150056224), combinations of lecithin and carbomer homopolymers (e.g.U.S. Pat. No. 6,676,958), and saponins, such as QUIL-A and QS21 (seee.g. Zhu D and W Tuo, 2016, Nat Prod Chem Res 3: e113(doi:10.4172/2329-6836.1000e113), optionally in combination with QS7(see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach(eds. Powell & Newman, Plenum Press, N Y, 1995); U.S. Pat. No.5,057,540). In some embodiments, the adjuvant is Freund's adjuvant(complete or incomplete). In certain embodiments, the adjuvant comprisesQUIL-A, such as for instance commercially obtainable from Brenntag (nowCroda) or Invivogen. QUIL-A contains the water-extractable fraction ofsaponins from the Quillaja saponaria Molina tree. These saponins belongto the group of triterpenoid saponins, that have a common triterpenoidbackbone structure. Saponins are known to induce a strong adjuvantresponse to T-dependent as well as T-independent antigens, as well asstrong cytotoxic CD8+ lymphocyte responses and potentiating the responseto mucosal antigens. They can also be combined with cholesterol andphospholipids, to form immunostimulatory complexes (ISCOMs), whereinQUIL-A adjuvant can activate both antibody-mediated and cell-mediatedimmune responses to a broad range of antigens from different origins. Incertain embodiments, the adjuvant is AS01, preferably AS01B. S01 is anAdjuvant System containing MPL (3-O-desacyl-4′-monophosphoryl lipid A),QS21 (Quillaja saponaria Molina, fraction 21) and liposomes. In certainembodiments, the AS01 is commercially available (GSK) or can be made asdescribed in WO 96/33739, incorporated herein by reference. Certainadjuvants comprise emulsions, which are mixtures of two immisciblefluids, e.g. oil and water, one of which is suspended as small dropsinside the other, and are stabilized by surface-active agents.Oil-in-water emulsions have water forming the continuous phase,surrounding small droplets of oil, while water-in-oil emulsions have oilforming the continuous phase. Certain emulsions comprise squalene (ametabolizable oil). Certain adjuvants comprise block copolymers, whichare copolymers formed when two monomers cluster together and form blocksof repeating units. An example of a water in oil emulsion comprising ablock copolymer, squalene and a microparticulate stabilizer isTiterMax®, which can be commercially obtained from Sigma-Aldrich.Optionally emulsions can be combined with or comprise furtherimmunostimulating components, such as a TLR4 agonist. Certain adjuvantsare oil in water emulsions (such as squalene or peanut oil) also used inMF59 (see e.g. EP0399843, U.S. Pat. Nos. 6,299,884, 6,451,325) and AS03,optionally in combination with immune stimulants, such as monophosphoryllipid A and/or QS21 such as in AS02 (see Stoute et al., 1997, N. Engl.J. Med. 336, 86-91). Further examples of adjuvants are liposomescontaining immune stimulants such as MPL and QS21 such as in AS01E andAS01B (e.g. US 2011/0206758). Other examples of adjuvants are CpG, andimidazoquinolines (such as imiquimod and R848). See, e.g., Reed G, etal., 2013, Nature Med, 19: 1597-1608.

In certain embodiments, the adjuvant comprises saponins, preferably thewater-extractable fraction of saponins obtained from Quillaja saponaria.In certain embodiments, the adjuvant comprises QS-21.

In certain embodiments, the adjuvant contains a toll-like receptor 4(TLR4) agonist. TLR4 agonists are well known in the art, see e.g. IretonG C and S G Reed, 2013, Expert Rev Vaccines 12: 793-807. In certainembodiments, the adjuvant is a TLR4 agonist comprising lipid A, or ananalog or derivative thereof.

The adjuvant, for example including a TLR4 agonist, may be formulated invarious ways, e.g. in emulsions such as water-in-oil (w/o) emulsions oroil-in-water (o/w) emulsions (examples are MF59, AS03), stable(nano-)emulsions (SE), lipid suspensions, liposomes, (polymeric)nanoparticles, virosomes, alum adsorbed, aqueous formulations (AF), andthe like, representing various delivery systems for immunomodulatorymolecules in the adjuvant and/or for the immunogens (see e.g. Reed etal, 2013, supra; Alving C R et al, 2012, Curr Opin Immunol 24: 310-315).

The immunostimulatory TLR4 agonist may optionally be combined with otherimmunomodulatory components, such as saponins (e.g. QUIL-A, QS7, QS21,Matrix M, Iscoms, Iscomatrix, etc), aluminum salts, activators for otherTLRs (e.g. imidazoquinolines, flagellin, CpG, dsRNA analogs, etc.), andthe like (see e.g. Reed et al, 2013, supra).

As used herein, the term “lipid A” refers to the hydrophobic lipidmoiety of an LPS molecule that comprises glucosamine and is linked toketo-deoxyoctulosonate in the inner core of the LPS molecule through aketosidic bond, which anchors the LPS molecule in the outer leaflet ofthe outer membrane of Gram-negative bacteria. For an overview of thesynthesis of LPS and lipid A structures, see, e.g., Raetz, 1993, J.Bacteriology 175:5745-5753, Raetz C R and C Whitfield, 2002, Annu RevBiochem 71: 635-700; U.S. Pat. Nos. 5,593,969 and 5,191,072. Lipid A, asused herein includes naturally occurring lipid A, mixtures, analogs,derivatives and precursors thereof. The term includes monosaccharides,e.g., the precursor of lipid A referred to as lipid X; disaccharidelipid A; hepta-acyl lipid A; hexa-acyl lipid A; penta-acyl lipid A;tetra-acyl lipid A, e.g., tetra-acyl precursor of lipid A, referred toas lipid IVA; dephosphorylated lipid A; monophosphoryl lipid A;diphosphoryl lipid A, such as lipid A from Escherichia coli andRhodobacter sphaeroides. Several immune activating lipid A structurescontain 6 acyl chains. Four primary acyl chains attached directly to theglucosamine sugars are 3-hydroxy acyl chains usually between 10 and 16carbons in length. Two additional acyl chains are often attached to the3-hydroxy groups of the primary acyl chains. E. coli lipid A, as anexample, typically has four C14 3-hydroxy acyl chains attached to thesugars and one C12 and one C14 attached to the 3-hydroxy groups of theprimary acyl chains at the 2′ and 3′ position, respectively.

As used herein, the term “lipid A analog or derivative” refers to amolecule that resembles the structure and immunological activity oflipid A, but that does not necessarily naturally occur in nature. LipidA analogs or derivatives may be modified to e.g. be shortened orcondensed, and/or to have their glucosamine residues substituted withanother amine sugar residue, e.g. galactosamine residues, to contain a2-deoxy-2-aminogluconate in place of the glucosamine-1-phosphate at thereducing end, to bear a galacturonic acid moiety instead of a phosphateat position 4′. Lipid A analogs or derivatives may be prepared fromlipid A isolated from a bacterium, e.g., by chemical derivation, orchemically synthesized, e.g. by first determining the structure of thepreferred lipid A and synthesizing analogs or derivatives thereof. LipidA analogs or derivatives are also useful as TLR4 agonist adjuvants (see,e.g. Gregg K A et al, 2017, MBio 8, eDD492-17, doi:10.1128/mBio.00492-17). For example, a lipid A analog or derivative canbe obtained by deacylation of a wild-type lipid A molecule, e.g., byalkali treatment. Lipid A analogs or derivatives can for instance beprepared from lipid A isolated from bacteria. Such molecules could alsobe chemically synthesized. Another example of lipid A analogs orderivatives are lipid A molecules isolated from bacterial cellsharboring mutations in, or deletions or insertions of enzymes involvedin lipid A biosynthesis and/or lipid A modification. MPL and 3D-MPL arelipid A analogs or derivatives that have been modified to attenuatelipid A toxicity. Lipid A, MPL and 3D-MPL have a sugar backbone ontowhich long fatty acid chains are attached, wherein the backbone containstwo 6-carbon sugars in glycosidic linkage, and a phosphoryl moiety atthe 4 position. Typically, five to eight long chain fatty acids (usually12-14 carbon atoms) are attached to the sugar backbone. Due toderivation of natural sources, MPL or 3D-MPL may be present as acomposite or mixture of a number of fatty acid substitution patterns,e.g. hepta-acyl, hexa-acyl, penta-acyl, etc., with varying fatty acidlengths. This is also true for some of the other lipid A analogs orderivatives described herein, however synthetic lipid A variants mayalso be defined and homogeneous. MPL and its manufacture are forinstance described in U.S. Pat. No. 4,436,727. 3D-MPL is for instancedescribed in U.S. Pat. No. 4,912,094B1, and differs from MPL byselective removal of the 3-hydroxymyristic acyl residue that is esterlinked to the reducing-end glucosamine at position 3 (compare forinstance the structure of MPL in column 1 vs 3D-MPL in column 6 of U.S.Pat. No. 4,912,094B1). In the art often 3D-MPL is used, while sometimesreferred to as MPL (e.g. the first structure in Table 1 of Ireton G Cand S G Reed, 2013, supra, refers to this structure as MPL®, butactually depicts the structure of 3D-MPL). Examples of lipid A (analogs,derivatives) according to the invention include MPL, 3D-MPL, RC529 (e.g.EP1385541), PET-lipid A, GLA (glycopyranosyl lipid adjuvant, a syntheticdisaccharide glycolipid; e.g. US20100310602, U.S. Pat. No. 8,722,064),SLA (e.g. Carter D et al, 2016, Clin Transl Immunology 5: e108 (doi:10.1038/cti.2016.63)), PHAD (phosphorylated hexaacyl disaccharide; thestructure of which is the same as that of GLA), 3D-PHAD,3D-(6-acyl)-PHAD (3D(6A)-PHAD) (PHAD, 3D-PHAD, and 3D(6A)PHAD aresynthetic lipid A variants, see e.g.avantilipids.com/divisions/adjuvants, which also provide structures ofthese molecules), E6020 (CAS Number 287180-63-6), 0N04007, OM-174, andthe like. For exemplary chemical structures of 3D-MPL, RC529, PET-lipidA, GLA/PHAD, E6020, 0N04007, and OM-174, see e.g. Table 1 in Ireton G Cand S G Reed, 2013, supra. For a structure of SLA, see e.g. FIG. 1 inReed S G et al, 2016, Curr Opin Immunol 41: 85-90. In certain preferredembodiments, the TLR4 agonist adjuvant comprises a lipid A analog orderivative chosen from 3D-MPL, GLA, or SLA.

Exemplary adjuvants comprising a lipid A analog or derivative includeGLA-LSQ (synthetic MPL [GLA], QS21, lipids formulated as liposomes),SLA-LSQ (synthetic MPL [SLA], QS21, lipids, formulated as liposomes),GLA-SE (synthetic MPL [GLA], squalene oil/water emulsion), SLA-SE(synthetic MPL [SLA], squalene oil/water emulsion), SLA-Nanoalum(synthetic MPL [SLA], aluminum salt), GLA-Nanoalum (synthetic MPL [GLA],aluminum salt), SLA-AF (synthetic MPL [SLA], aqueous suspension), GLA-AF(synthetic MPL [GLA], aqueous suspension), SLA-alum (synthetic MPL[SLA], aluminum salt), GLA-alum (synthetic MPL [GLA], aluminum salt),and several of the GSK ASxx series of adjuvants, including AS01 (MPL,QS21, liposomes), AS02 (MPL, QS21, oil/water emulsion), AS25 (MPL,oil/water emulsion), AS04 (MPL, aluminum salt), and AS15 (MPL, QS21,CpG, liposomes). See, e.g., WO 2013/119856, WO 2006/116423, U.S. Pat.Nos. 4,987,237, 4,436,727, 4,877,611, 4,866,034, 4,912,094, 4,987,237,U.S. Pat. Nos. 5,191,072, 5,593,969, 6,759,241, 9,017,698, 9,149,521,9,149,522, 9,415,097, 9,415,101, 9,504,743, Reed G, et al., 2013, supra,Johnson et al., 1999, J Med Chem, 42:4640-4649, and Ulrich and Myers,1995, Vaccine Design: The Subunit and Adjuvant Approach; Powell andNewman, Eds.; Plenum: New York, 495-524.

Non-glycolipid molecules may also be used as TLR4 agonist adjuvants,e.g. synthetic molecules such as Neoseptin-3 or natural molecules suchas LeIF, see e.g. Reed S G et al, 2016, supra.

In another aspect the invention relates to a FimH(F71mut) orFimH(F71mut/F144mut) polypeptide of the invention, a polynucleotideencoding a FimH(F71mut) or FimH(F71mut/F144mut) polypeptide of theinvention or a pharmaceutical composition of the invention for use as amedicament.

In a further aspect the invention relates to the use of a FimH(F71mut)or FimH(F71mut/F144mut) polypeptide of the invention, a polynucleotideencoding a FimH(F71mut) or FimH(F71mut/F144mut) polypeptide of theinvention or a pharmaceutical composition described herein as amedicament for inducing an immune response against a gram negativebacterium of the family of Enterobacteriaceae.

As used herein the terms “immunogen” or “immunogenic” or “antigen” areused interchangeably to describe a molecule capable of inducing animmunological response against itself on administration to a recipient,either alone, in conjunction with an adjuvant, or presented on a displayvehicle.

As used herein, an “immunological response” or “immune response” to anantigen or composition refers to the development in a subject of ahumoral and/or a cellular immune response to the antigen or an antigenpresent in the composition.

In a further aspect, the invention relates a FimH(F71mut) orFimH(F71mut/F144mut) polypeptide of the invention, a polynucleotideencoding a FimH(F71mut) or FimH(F71mut/F144mut) polypeptide of theinvention or a pharmaceutical composition described herein for use ininducing an immune response against a bacterial infection caused a gramnegative bacterium of the family Enterobacteriaceae. In certainembodiments the bacterial infection is caused by Klebsiella spp., or E.coli. In a preferred embodiment, the bacterial infection is caused by E.coli. Thus in one embodiment, the invention relates to the use of aFimH(F71mut) or FimH(F71mut/F144mut) polypeptide of the invention aFimH(F71mut) or FimH(F71mut/F144mut) polypeptide of the invention or apharmaceutical composition described herein as a medicament for inducingan immune response against E. coli or Klebsiella, preferably E. coli.

In preferred embodiments, the bacterial infection, caused by agram-negative bacterium of the family Enterobacteriaceae is an infectionby E. coli, e.g. by ExPEC, for instance the infection can be a urinarytract infection (UTI). In one embodiment, the invention relates to thepolypeptide comprising a FimH lectin domain as described herein, apolynucleotide as described or a pharmaceutical composition describedherein for use in treating, preventing, or suppressing symptoms and/orsequelae associated with a UTI in a subject. In certain embodiments,said UTI is a rUTI. E. coli is one of the main causative agents of UTIsand rUTIs which are an important health care problem in young femalesand older adults. Thus, in preferred embodiments, the bacterialinfection is a UTI or rUTI caused by E. coli.

In one embodiment, the invention relates to a method of treating,preventing, or suppressing symptoms and/or sequelae associated with anenterobacillus-related condition a subject in need thereof. The methodcomprises administering to the subject an effective amount of aFimH(F71mut) or FimH(F71mut/F144mut) polypeptide of the invention, apolynucleotide encoding a FimH(F71mut) or FimH(F71mut/F144mut)polypeptide of the invention or a pharmaceutical composition describedherein. Preferably, the administration induces an immune response thatis effective in treating or preventing an enterobacillus-relatedcondition. Preferably, the enterobacillus-related condition is aurogenital tract infection, more particularly a UTI or rUTI.

The invention also relates to use of a FimH(F71mut) orFimH(F71mut/F144mut) polypeptide of the invention, a polynucleotideencoding a FimH(F71mut) or FimH(F71mut/F144mut) polypeptide of theinvention or a pharmaceutical composition described herein for themanufacture of a medicament for treating, preventing, or suppressing abacterial infection caused a gram negative bacterium of the familyEnterobacteriaceae, preferably a bacterial infection caused by E. coli.More preferably, the bacterial infection is a UTI or recurrent UTI(rUTI) caused by E. coli.

The invention further relates to a method for the production of apolypeptide of the invention, the method comprising culturing arecombinant cell containing the polynucleotide encoding a FimH(F71mut)or FimH(F71mut/F144mut) polypeptide of the invention and/or the vectoras described herein, wherein the culturing takes place under conditionsconducive to the production of the polypeptide.

In certain embodiments, the method further comprises recovering thepolypeptide, which is optionally followed by formulation into apharmaceutical composition.

Preferably an E. coli cell, for example an E. coli BL21 derivative, cellis used in the method for producing a FimH(F71mut) orFimH(F71mut/F144mut) polypeptide of the invention.

The recovery of the polypeptide preferably includes a purificationand/or isolation step which can be performed using conventional proteinpurification methods well known in the art. Such methods may for exampleinclude ammonium sulfate or ethanol precipitation, acid extraction,anion and/or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography, and/or lectinchromatography.

Typical examples for such purification and/or isolation may utilize anantibody to the protein or to a His tag or cleavable leader or tail thatis expressing as part of the protein structure. In certain embodiments,the polypeptide described herein have a His-tag included and arepurified by methods such as IMAC affinity purification. In certainembodiments, the polypeptides described herein do not comprise aHis-tag, in such cases purification can be performed by chromatography,for example ion exchange chromatography (IEX), hydrophobic interactionchromatography (HIC) and/or size exclusion chromatography.

Definitions

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms citedherein have the meanings as set in the specification. All patents,published patent applications and publications cited herein areincorporated by reference as if set forth fully herein. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

Throughout this description and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.Any of the aforementioned terms of “comprising”, “containing”,“including”, and “having”, whenever used herein in the context of anaspect or embodiment of the invention can be replaced with the term“consisting of” or “consisting essentially of” to vary scopes of thedisclosure.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or”, afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or.”

As used herein, the term “pharmaceutically acceptable carrier” refers toa non-toxic material that does not interfere with the effectiveness of acomposition according to the invention or the biological activity of acomposition according to the invention. A “pharmaceutically acceptablecarrier” can include any excipient, diluent, filler, salt, buffer,stabilizer, solubilizer, oil, lipid, lipid containing vesicle,microsphere, liposomal encapsulation, or other material well known inthe art for use in pharmaceutical formulations. It will be understoodthat the characteristics of the pharmaceutically acceptable carrier willdepend on the route of administration for a particular application.According to particular embodiments, in view of the present disclosure,any pharmaceutically acceptable carrier suitable for use in a vaccinecan be used in the invention. Suitable excipients include but are notlimited to sterile water, saline, dextrose, glycerol, ethanol, or thelike and combinations thereof, as well as stabilizers, e.g. Human SerumAlbumin (HSA) or other suitable proteins and reducing sugars.

As used herein, the term “effective amount” refers to an amount of anactive ingredient or component that elicits the desired biological ormedicinal response in a subject. An effective amount can be determinedempirically and in a routine manner, in relation to the stated purpose.For example, in vitro assays can optionally be employed to help identifyoptimal dosage ranges.

As used herein, “subject” or “patient” means any animal, preferably amammal, most preferably a human, who will be or has been vaccinated by amethod or composition according to an embodiment of the invention. Theterm “mammal” as used herein, encompasses any mammal. Examples ofmammals include, but are not limited to, cows, horses, sheep, pigs,cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.,most preferably a human. In certain embodiments, a subject is a humanadult. As used herein, the term “human adult” refers to a human that is18 years or older. In certain embodiments, a subject is less than 18years old, e.g. 0-18 years old, e.g. 9-18 years old, or 12-18 years old.In certain embodiments, a subject is a human subject of about 18 toabout 50 years. In certain embodiments, a subject is a human of about 50to about 100 years, e.g. 50-85 years, 60-80 years, 50 years or older, 55years or older, 60 years or older, 65 years or older, 70 years or older,75 years or older, 80 years or older, 85 years or older. In someembodiments hereof the subject is not older than 85 years, not olderthan 80 years, not older than 75 years. In certain embodiments, a humansubject is a male. In certain embodiments, a human subject is a female.

As used herein, a “UTI” means an infection of the kidney, bladder,ureter, or urethra. Symptoms of UTI may include one or more of burningfeeling when urinating, frequent or intense urge to urinate, incompletebladder emptying, urine having abnormal look and/or smell, elevatedwhite blood cells in urine, feeling tired or shaky, feeling disoriented,fever or chills, malaise, pain or pressure in back, lower abdomen,pelvis or bladder. However, in some patients symptoms may be absent ornon-specific. Sequelae of UTI may include systemic complications such asinvasive disease and sepsis. In certain embodiments, a UTI is clinicallyand/or microbiologically documented, e.g. confirmed with bacterialculture of urine and/or with molecular or other methods. In certainembodiments, the subject is a human subject that previously has had orcurrently is having a UTI. In certain embodiments, the subject has had aUTI within the last two years, the last year, or the last 6 months. Incertain embodiments, the subject has had or currently has a recurrentUTI (rUTI). A “rUTI” as used herein means at least two infections in sixmonths or at least three UTIs in one year. In certain embodiments, asubject to whom the FimH(F71mut) or FimH(F71mut/F144mut), the FimCHcomplex of the invention, a fusion polypeptide of the invention, or acomposition of the invention is administered, has suffered at least twoUTIs within the last two years, within the last year, or within the lastsix months. In certain embodiments, the subject has suffered fromcomplicated UTI. A ‘complicated UTI’ as used herein means a UTIassociated with a condition, such as structural or functionalabnormalities of the genitourinary tract or the presence of anunderlying disease. In certain embodiments, a UTI leads to elevatednumbers of white blood cells in urine or other urine abnormalities. Incertain embodiments, a subject with UTI has a number of bacteria inurine, i.e. the urine is not sterile, e.g. a bacterial cell count of atleast about 10 cells/mL, at least about 100 cells/mL, at least about 10³cells/mL, e.g. at least about 10⁴ cells/mL, e.g. at least about 10⁵cells/mL.

As used herein, an “immunological response” or “immune response” to anantigen or composition refers to the development in a subject of ahumoral and/or a cellular immune response to the antigen or an antigenpresent in the composition.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.

The word “about” or “approximately” when used in association with anumerical value (e.g. about 10) preferably means that the value may bethe given value (of 10) more or less 10%, preferably more or less 5% ofthe value.

The terms “homology”, “sequence identity” and the like are usedinterchangeably herein. Sequence identity is herein defined as arelationship between two or more amino acid (polypeptide or protein)sequences or two or more nucleic acid (polynucleotide) sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between amino acid or nucleic acidsequences, as the case may be, as determined by the match betweenstrings of such sequences. “Similarity” between two amino acid sequencesis determined by comparing the amino acid sequence and its conservedamino acid substitutes of one polypeptide to the sequence of a secondpolypeptide. “Identity” and “similarity” can be readily calculated byknown methods.

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms, depending on the length of the twosequences. Sequences of similar lengths are preferably aligned using aglobal alignment algorithm (e.g. Needleman Wunsch) which aligns thesequences optimally over the entire length, while sequences ofsubstantially different lengths are preferably aligned using a localalignment algorithm (e.g. Smith Waterman). Sequences may then bereferred to as “substantially identical” or “essentially similar” whenthey (when optimally aligned by for example the programs GAP or BESTFITusing default parameters) share at least a certain minimal percentage ofsequence identity (as defined below). GAP uses the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength (full length), maximizing the number of matches and minimizingthe number of gaps. A global alignment is suitably used to determinesequence identity when the two sequences have similar lengths.Generally, the GAP default parameters are used, with a gap creationpenalty=50 (nucleotides)/8 (proteins) and gap extension penalty=3(nucleotides)/2 (proteins). For nucleotides the default scoring matrixused is nwsgapdna and for proteins the default scoring matrix isBlosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequencealignments and scores for percentage sequence identity may be determinedusing computer programs, such as the GCG Wisconsin Package, Version10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego,Calif. 92121-3752 USA, or using open source software, such as theprogram “needle” (using the global Needleman Wunsch algorithm) or“water” (using the local Smith Waterman algorithm) in EmbossWIN version2.10.0, using the same parameters as for GAP above, or using the defaultsettings (both for ‘needle’ and for ‘water’ and both for protein and forDNA alignments, the default Gap opening penalty is 10.0 and the defaultgap extension penalty is 0.5; default scoring matrices are Blosum62 forproteins and DNAFull for DNA). When sequences have a substantiallydifferent overall lengths, local alignments, such as those using theSmith Waterman algorithm, are preferred.

Alternatively, percentage similarity or identity may be determined bysearching against public databases, using algorithms such as FASTA,BLAST, etc. Thus, the nucleic acid and protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify other family membersor related sequences. Such searches can be performed using the BLASTnand BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol.Biol. 215:403-10. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTx program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., BLASTx and BLASTn) can be used. See the homepage of the NationalCenter for Biotechnology Information at hypertext transferprotocol://www.ncbi.nlm.nih.gov/.

DESCRIPTION OF THE SEQUENCES

TABLE 1 Sequences Description SEQUENCE SEQ ID NO. FimH_(LD) sequenceFACKTANGTAIPIGGGSANVYVNLAPAVNVGQNLVVDLSTQIF 1 (FimH_(LD) 23-10)CHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTG FimHt sequenceFACKTANGTAIPIGGGSANVYVNLAPAVNVGQNLVVDLSTQIF 2CHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYAR TGGQVTAGNVQSIIGVTFVYQFimC full length GVALGATRVIYPAGQKQVQLAVTNNDENSTYLIQSWVENADGV 3KDGRFIVTPPLFAMKGKKENTLRILDATNNQLPQDRESLFWMNVKAIPSMDKSKLTENTLQLAIISRIKLYYRPAKLALPPDQAAEKLRFRRSANSLTLINPTPYYLTVTELNAGARVLENALVPPMGESTVKLPSDAGSNITYRTINDYGALTPKMTGVME Example of FimHMKRVITLFAVLLMGWSVNAWSFACKTANGTAIPIGGGSANVYV 4 23-10 (full length)NLAPAVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYG sequenceGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ SEQ ID NO: 2 ofMKRVITLFAVLLMGWSVNAWSFACKTANGTAIPIGGGSANVYV 5 U.S. Pat. No. 6,500,434NLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYG (example fullGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALY length FimHLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYAN sequence)NDVVVPTGGCDVSARDVTVTLPDYRGSVPIPLTVYCAKSQNLGYYLSGTHADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVTQ SEQ ID NO: 29 ofFACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIF 6 U.S. Pat. No. 6,737,063CHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKYSGSSYPFPT (example FimHTSETPRVVYNSRTDKPWPVALYLTPVSSAGGLVIKAGSLIAVL sequence withILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTL truncation at N-PDYRGSVPIPLTVYCAKSQNLGYYLSGTHADAGNSIFTNTASF terminus)SPAQGVGVQLTRNGTIIPTNNTVSLGAVGTSAVSLGLTANYAR TGGQVTAGNVQSIIGVTFVYQExample of a FACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIF 7FimH_(LD) sequence CHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGLVIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGG

Other examples of sequences for FimH polypeptides are described in U.S.Pat. No. 6,737,063, for example any one of SEQ ID NO: 23-45 or 55therein, and these are all incorporated by reference herein.

EXAMPLES

The following examples of the invention are to further illustrate thenature of the invention. It should be understood that the followingexamples do not limit the invention and that the scope of the inventionis to be determined by the appended claims.

To understand the impact of FimH conformational changes in the vaccineefficacy, several variants of FimH that contain different mutations thatcould potentially lock the protein in the low affinity status weredesigned, aiming to identify improved FimH variants that inducefunctional antibodies capable of reducing bacterial adhesion andcolonization of the bladder. To optimize the chances of finding asuitable FimH lectin domain variant, variants with a different predictedmode of action were selected. The previously described mutantsFimH_Q133K and FimH_R60P as well as a wild-type (WT) FimH were takenalong as controls.

Example 1: Ability to Induce Inhibiting Antibodies

It has been shown that antibodies generated against FimH in the lowaffinity conformation are capable of blocking the bacterial cell andreducing colony formation in the bladder. Therefore as a first step, weevaluated the functionality of antibodies induced by the differentvariants of FimH locked in the low affinity conformation by using anadhesion inhibition assay (AIA).

Materials and Methods

FimH Design and Expression

FimC and FimH were expressed in a pET-DUET or pET-22b vector usingheterologous signal sequences for expression in the periplasm and aC-terminal His-tag on FimC for affinity purification using immobilizedmetal affinity chromatography (IMAC). Expression was induced using IPTGand protein was extracted and purified using IMAC purification (Talon).

Immunization

Wistar rats received 4 intramuscular (i.m.) immunizations at days 0, 7,10 and 18 with the different FimH variants (60 μg each variant/dose)combined with a non-Freund adjuvant (Speedy rat 28-Day model,Eurogentec). Functionality of serum antibodies was investigated at day 0(pre-immunization) and day 28 (post-immunization) by Adhesion InhibitionAssay (AIA) as described below.

Adhesion Inhibition Assay (AIA)

Bacteria (E. coli strain J96) were labeled with a fluoresceinisothiocyanate (FITC). Labeled bacteria were incubated with bladderurothelial cells (5637 cell line) for 1 h at 37° C. The % of adherentbacteria was measured by flow cytometry. For evaluation of seruminhibition, bacteria were previously incubated with serum samples for 30minutes at 37° C. and then mixed with 5637 cells.

ELISA

96-well plates were coated overnight with 1 ug/mL of FimH. Afterwashing, coated wells were incubated with blocking buffer[phosphate-buffered saline (PBS)+2% bovine serum albumin (BSA)] for 1hour at room temperature. After washing with PBS+0.05% TWEEN 20, serumwas added to the plates that were then incubated for 1 hour at roomtemperature. After washing, goat anti-rat antibody conjugated tohorseradish peroxidase diluted in PBS with 2% BSA was added to each wellfor 1 hour at room temperature. After a final washing, the reaction wasdeveloped with tetramethylbenzidine substrate. The reaction was stoppedwith 1M phosphoric acid, and absorbance is measured at 450 nm.

Results

Serum antibody inhibitory titers were calculated as half maximalinhibitory concentration (IC50) based on a 4-parameter logistic (4PL)regression model. In addition, levels of serum antibodies induced bydifferent FimH variants (total IgG) were evaluated by ELISA. IC50titers, defined as half maximal effective concentration, were calculatedbased on duplicate 6-step titration curves that were analyzed with a 4PLnonlinear regression model.

In a preliminary experiment, several different FimH variants were testedfor their ability to induce inhibitory antibodies. As can be seen inFIG. 1A, the variants FimH(F71Y), FimH(F144V) induced the highest levelsof functional antibodies. The FimH(F144V) variant was previouslydescribed in EP patent application number 20152217, filed in the name ofJanssen Pharmaceuticals, Inc on 16 Jan. 2020, incorporated by referenceherein. However, it was surprising and entirely unpredictable prior tothe present invention that FimH(F71Y) would induce similarly high levelsof inhibitory antibodies. To confirm the preliminary results, the AIAassay was repeated with a larger number of animals, in this experimentit was confirmed that both FimH(F71Y) and FimH(F144V) were reliablycapable of inducing high levels of inhibitory antibodies (FIG. 1B).

The FimH(F71Y) and FimH(F144V) variants appear to be locked in the lowaffinity conformation by different mechanisms that induce aconformational change of the binding pocket. The substitution in theFimH(F71Y) variant prevents the residue to fold into a hydrophobicpocket (relaxed state) making the tense state more favorable whereas inthe FimH(F144V) variant, the substitution is situated relatively closeto the mannose binding pocket which stabilizes the low affinityconformation. Since the conformational change to the low affinity statesis brought about by these two distinct mechanisms, we hypothesized thata lectin domain comprising both the F71Y and the F144V substitutioncould be even more stably locked in the low affinity conformation whichwould provide an extra advantage to a FimH variant of that kind. To testthis hypothesis, we created a FimH double mutant which comprises boththe F71Y and the F144V substitution it its lectin domain(FimH(F71Y/F144V)). As can be seen in FIG. 1B, FimH(F71Y/F144V) wasequally capable of inducing high levels of inhibitory antibodies.

Based on these results, FimH(F71Y) and FimH(F71Y/F144V) were selected asthe lead candidates. Their characteristics were further analyzed asdescribed below.

Example 2: Design Novel Variants—SPR Data

SPR

To gain a detailed insight into the binding affinity and kinetics of theinteraction of FimH lectin domain variants with then-Heptyl-α-D-mannopyranoside ligand, surface plasmon resonance (SPR)measurements were performed. In brief, the FimH variants were titratedinto the Mannoside ligand 5 surface (Rabbani S et al, J Biol. Chem.,2018, 293(5):1835-1849) with the top concentrations of either 3 or 10 μMin HBS-N (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactantP20). The proteins were injected from 0.12 to 10 μM or 0.036 to 3.0 μMusing a single cycle kinetics injection.

Results

Previously described mutants FimH_Q133K and FimH_R60P have a mutation inthe mannose interacting residues in the binding pocket. These mutationsare predicted to directly influence the binding interaction withmannose. These mutants were taken as positive controls. The doublemutant R60P_Q133K was taken along to check for potentially enhancedeffects. Additionally, wild-type (WT) FimH lectin domain was taken alongas a negative control.

The variants affinity for binding to mannose was assessed using SPR. Theresults are presented in table 2. As expected, the lectin domainvariants having mutations in the mannoside binding pocket of FimH (Q133Kand R60P_Q133K) did not bind to mannoside at all. The R60P mutant showedlow affinity for mannoside, as expected.

Despite being capable of inducing high levels inhibitory antibodies, thevariant comprising the F71Y substitution still showed some affinity formannoside, indicating that this mutation did not completely abolishbinding to mannoside. In the FimH lectin domain variants comprising theF144V substitution the binding to mannoside was completely abolished (aspreviously disclosed for the single F144V mutant in EP patentapplication number 20152217, filed in the name of JanssenPharmaceuticals, Inc on 16 Jan. 2020, incorporated by reference herein).In the mutant comprising both F71Y and F144V binding to mannoside wasalso completely abolished.

TABLE 2 Affinity measurements of FimH_(LD) variants to mannoside VariantAffinity* F144V No binding F71Y Low affinity F144V/F71Y No binding R60PLow affinity R60P-Q133K No binding Q133K No binding FimH_LD wt Highaffinity *Low affinity K_(D) ≥ 1000 nM; Medium affinity K_(D) between100-1000 nM; High affinity K_(D) ≤ 100 nM

Example 3: Ability to Bind Characterized Inhibitory mAbs 475 and 926

Mutations in the lectin domain of FimH may cause the loss of epitopesthat are crucial in eliciting a strong and functional immune responsesuch as the epitopes that are present in the binding pocket of FimH. Toensure that the integrity of the binding pocket was not compromised bythe mutations described herein, the binding of monoclonal antibodies(mAb) mAb475 and mAb926 to the mutant FimH lectin domains was assessed.mAb475 and mAb926, recognize overlapping but distinct epitopes on theFimH lectin domain within the mannose-binding pocket of the FimH(Kisiela et al. 2013 & 2015).

Results

Mutated FimH lectin domains have been previously described inWO02102974. WO02102974 describes an extensive list of possible mutationsof mostly unspecified mutations (65 possible mutation sites aresuggested in the lectin domain of FimH which is approximately 159 aminoacids long). However, WO02102974 indicates that the FimH lectin domainshaving an amino acid substitution at position 54, 133 or 135 are themost promising candidates, FimH_Q133K is explicitly mentioned as ahighly preferred option. It was therefore quite surprising thatFimH-23-10_Q133K was not recognized by the functional mAb475, indicatingintegrity issues of the binding pocket (Table 3). In contrast, bothFimH(F71Y) and FimH(F144V) were recognized by both mAb475 and mAb926indicating that the binding pocket remained completely intact (Table 3).Also the FimH(F71Y/F144V) double mutant was still recognized by bothantibodies, indicating that the binding pocket also remained intact inthis double mutant (Table 3).

TABLE 3 Ability to bind characterized inhibitory mAbs 475 and 926Binding pocket integrity Variant mAb 475 mAb 926 F144V Binding BindingF71Y Binding Binding F71Y/F144V Binding Binding R60P Binding BindingR60P-Q133K No binding Binding Q133K No binding Binding FimH_LD wtBinding Binding

Example 4: Conformational State Analysis of the FimH Lectin DomainVariants by NMR

¹H ¹⁵N heteronuclear single quantum coherence nuclear magnetic resonance(HSQC NMR) spectra of uniformly ¹⁵N-labeled FimH_(LD) variants, producedas described in the method section with the addition of ¹⁵N to thegrowth media, were measured in the absence and presence of then-Heptyl-α-D-mannopyranoside ligand to assess structural differences ona residue-based level. Protein was concentrated to 150 μM and measuredin 20 mM phosphate buffer, pH 7.4, with 7%-10% D₂O. Then-Heptyl-α-D-mannopyranoside ligand was dissolved in D₂O and addedstepwise to the protein up to a 10-fold molar excess. The spectra ofeach sample were compared to the publically available reference spectra(Rabbani S et al, J Biol. Chem., 2018, 293(5):1835-1849).

Results

FimH variants FimH(F71Y), FimH(F144V) and FimH(F71Y/F144V) were analyzedfor their ability to remain in the low affinity confirmation in thepresence of the mannoside ligand. In the absence of ligand, all threevariants are in a low affinity confirmation when comparing chemicalshifts of residues known to be indicative of the high affinity state(Rabbani S et al, J Biol. Chem., 2018, 293(5):1835-1849). In contrast,the FimH_(LD) 23-10 wild type is locked in the high affinityconformation in absence of the mannoside ligand. However in the presenceof ligand, FimH(F71Y) and FimH(F144V) switch back to the high affinityconfirmation, while FimH (F71Y/F144V) stays in the low affinityconfirmation, e.g. no chemical shift is observed after addition ofligand to the FimH(F71Y/F144V) (FIG. 2 and Table 4).

TABLE 4 Tabular representation of the chemical shifts of selected aminoacid residues as measured by NMR. 23-10 R60P F144V F71Y F144V/F71YResidue Apo Ligand Apo Ligand Apo Ligand Apo Ligand Apo Ligand 1.Unknown (specific L H L H L H L H L L for FimH_(LD) 23-10) 2. Unknown(specific L H L H L H L H L L for FimH_(LD) 23-10) Ala (A) residue 2 H HL H L H L H L L Ala (K) position 4 H H L H L H L H L L Asp (D) residue54 H H L H L H L H L L Gln (Q) residue 133 H H L H L H L H L L Asp (D)residue 141 H H L H L H L H L L

Apart from two unknown residues that differ from the K12 wild-type, theFimH_(LD) 23-10 wild type is considered to be already in the highaffinity (H) conformation without ligand. The single mutant variantsF71Y and F144V are in the low affinity (L) conformation without ligandbut switch to H conformation in the presence of ligand, while the doublemutant variant F71Y/F144V remained in the L conformation even in thepresence of ligand. This inability to switch back to the high affinityconformation makes the FimH(F71Y/F144V) especially attractive for use inpharmaceutical compositions of vaccines, as it for instance ensures thatno additional quality or stability controls are needed to test whetherthe conformation is stable during storage or under other storageconditions. Additionally, the inability to switch back into the highaffinity conformation allows the use of the FimH(F71Y/F144V) as aresearch tool to study the mechanism of action or to gain conformationalknowledge and it allows the FimH(F71Y/F144V) to be used to generatespecific antibodies with useful research, diagnostic and possiblypharmaceutical applications.

Thus, in conclusion, of all of the FimH variants tested, FimH(F71Y),FimH(F144V), and FimH(F71Y/F144V) are capable of inducing the highestlevels of functional inhibitory antibodies while keeping the bindingpocket of FimH intact. FimH(F71Y/F144V) has the additional advantages ofcompletely abolishing the binding to mannoside and of remaining lockedin the low affinity conformation even in the presence of the ligand.

The invention claimed is:
 1. A polypeptide comprising a FimH lectindomain comprising Tyrosine (Y) or Tryptophan (W) at the position thatcorresponds to position 71 in SEQ ID NO: 1, wherein the FimH lectindomain has an amino acid sequence having at least 90% sequence identitywith SEQ ID NO:
 1. 2. The polypeptide according to claim 1, wherein theFimH lectin domain comprises Tyrosine (Y) at the position thatcorresponds to position 71 in SEQ ID NO:
 1. 3. The polypeptide accordingto claim 1, wherein the polypeptide further comprises an amino acidother than phenylalanine (F) at a position corresponding to position 144in the amino acid sequence of SEQ ID NO:
 1. 4. The polypeptide accordingto claim 3, wherein the polypeptide comprises an amino acid selectedfrom the group consisting of valine (V), isoleucine (I), leucine (L),glycine (G), methionine (M), and alanine (A) at the position thatcorresponds to position 144 in SEQ ID NO:
 1. 5. The polypeptideaccording to claim 3, wherein the FimH lectin domain comprises Valine(V) at the position that corresponds to position 144 in SEQ ID NO:
 1. 6.The polypeptide according to claim 1, wherein the FimH lectin domain hasan amino acid sequence having at least 95% sequence identity with SEQ IDNO:
 1. 7. The polypeptide according to claim 6, wherein the FimH lectindomain comprises SEQ ID NO: 1 wherein the phenylalanine residue atposition 71 is substituted by tyrosine and wherein the phenylalanineresidue at position 144 is substituted by valine.
 8. The polypeptideaccording to claim 1, wherein the polypeptide further comprises a FimHpilin domain.
 9. The polypeptide according to claim 6, wherein thepolypeptide is a full length FimH having at least 90% sequence identitywith SEQ ID NO:
 2. 10. A pharmaceutical composition comprising thepolypeptide according to claim
 1. 11. The polypeptide of claim 6,wherein the FimH lectin domain comprises SEQ ID NO: 1 wherein thephenylalanine residue at position 71 is substituted by tyrosine.
 12. Thepolypeptide of claim 9, wherein the FimH lectin domain comprises SEQ IDNO: 2 wherein the phenylalanine residue at position 71 is substituted bytyrosine and the phenylalanine residue at position 144 is substituted byvaline.
 13. A pharmaceutical composition comprising the polypeptide ofclaim
 2. 14. A pharmaceutical composition comprising the polypeptide ofclaim
 3. 15. A pharmaceutical composition comprising the polypeptide ofclaim
 4. 16. A pharmaceutical composition comprising the polypeptide ofclaim
 5. 17. A pharmaceutical composition comprising the polypeptide ofclaim
 6. 18. A pharmaceutical composition comprising the polypeptide ofclaim
 7. 19. A pharmaceutical composition comprising the polypeptide ofclaim
 8. 20. A pharmaceutical composition comprising the polypeptide ofclaim
 9. 21. A pharmaceutical composition comprising the polypeptide ofclaim
 11. 22. A pharmaceutical composition comprising the polypeptide ofclaim
 12. 23. The pharmaceutical composition of claim 13, furthercomprising an adjuvant.
 24. The pharmaceutical composition of claim 14,further comprising an adjuvant.
 25. The pharmaceutical composition ofclaim 15, further comprising an adjuvant.
 26. The pharmaceuticalcomposition of claim 16, further comprising an adjuvant.
 27. Thepharmaceutical composition of claim 17, further comprising an adjuvant.28. The pharmaceutical composition of claim 18, further comprising anadjuvant.
 29. The pharmaceutical composition of claim 19, furthercomprising an adjuvant.
 30. The pharmaceutical composition of claim 20,further comprising an adjuvant.
 31. The pharmaceutical composition ofclaim 21, further comprising an adjuvant.
 32. The pharmaceuticalcomposition of claim 22, further comprising an adjuvant.
 33. Thepharmaceutical composition of claim 23, further comprising an adjuvant.